The epileptic encephalopathies are a group of highly heterogeneous genetic disorders. The majority of disease-causing mutations alter genes encoding voltage-gated ion channels, neurotransmitter receptors, or synaptic proteins. We have identified a novel de novo pathogenic K+ channel variant in an idiopathic epileptic encephalopathy family. Here, we report the effects of this mutation on channel function and heterologous expression in cell lines. We present a case report of infantile epileptic encephalopathy in a young girl, and trio-exome sequencing to determine the genetic etiology of her disorder. The patient was heterozygous for a de novo missense variant in the coding region of the KCNB1 gene, c.1133T>C. The variant encodes a V378A mutation in the α subunit of the Kv2.1 voltage-gated K+ channel, which is expressed at high levels in central neurons and is an important regulator of neuronal excitability. We found that expression of the V378A variant results in voltage-activated currents that are sensitive to the selective Kv2 channel blocker guangxitoxin-1E. These voltage-activated Kv2.1 V378A currents were nonselective among monovalent cations. Striking cell background-dependent differences in expression and subcellular localization of the V378A mutation were observed in heterologous cells. Further, coexpression of V378A subunits and wild-type Kv2.1 subunits reciprocally affects their respective trafficking characteristics. A recent study reported epileptic encephalopathy-linked missense variants that render Kv2.1 a tonically activated, nonselective cation channel that is not voltage activated. Our findings strengthen the correlation between mutations that result in loss of Kv2.1 ion selectivity and development of epileptic encephalopathy. However, the strong voltage sensitivity of currents from the V378A mutant indicates that the loss of voltage-sensitive gating seen in all other reported disease mutants is not required for an epileptic encephalopathy phenotype. In addition to electrophysiological differences, we suggest that defects in expression and subcellular localization of Kv2.1 V378A channels could contribute to the pathophysiology of this KCNB1 variant.

The Kv2.1 voltage-gated K+ channel is widely expressed throughout mammalian brain, where it contributes to dynamic activity-dependent regulation of intrinsic neuronal excitability. Here we show that somatic plasma membrane Kv2.1 clusters are juxtaposed to clusters of intracellular ryanodine receptor (RyR) Ca2+ -release channels in mouse brain neurons, most prominently in medium spiny neurons (MSNs) of the striatum. Electron microscopy-immunogold labeling shows that in MSNs, plasma membrane Kv2.1 clusters are adjacent to subsurface cisternae, placing Kv2.1 in close proximity to sites of RyR-mediated Ca2+ release. Immunofluorescence labeling in transgenic mice expressing green fluorescent protein in specific MSN populations reveals the most prominent juxtaposed Kv2.1:RyR clusters in indirect pathway MSNs. Kv2.1 in both direct and indirect pathway MSNs exhibits markedly lower levels of labeling with phosphospecific antibodies directed against the S453, S563, and S603 phosphorylation site compared with levels observed in neocortical neurons, although labeling for Kv2.1 phosphorylation at S563 was significantly lower in indirect pathway MSNs compared with those in the direct pathway. Finally, acute stimulation of RyRs in heterologous cells causes a rapid hyperpolarizing shift in the voltage dependence of activation of Kv2.1, typical of Ca2+ /calcineurin-dependent Kv2.1 dephosphorylation. Together, these studies reveal that striatal MSNs are distinct in their expression of clustered Kv2.1 at plasma membrane sites juxtaposed to intracellular RyRs, as well as in Kv2.1 phosphorylation state. Differences in Kv2.1 expression and phosphorylation between MSNs in direct and indirect pathways provide a cell- and circuit-specific mechanism for coupling intracellular Ca2+ release to phosphorylation-dependent regulation of Kv2.1 to dynamically impact intrinsic excitability.

Antibodies specific for the drk1 polypeptide were used to characterize the corresponding protein in rat brain. Recombinant and synthetic immunogens containing fragments of the drk1 polypeptide were produced. Antibodies raised to these immunogens display monospecific reactions with the same 130-kDa polypeptide on immunoblots of adult rat brain membranes. Immunoprecipitation of 125I-labeled brain membranes identifies a 38-kDa peptide in tight association with the drk1 polypeptide. Immunohistochemical staining of sections of adult rat cortex shows that drk1 protein is restricted to neurons, where staining is present on dendrites and cell bodies but not on axons. These studies point to the value of such immunological reagents to the further characterization of the components of this delayed rectifier K+ channel in the mammalian central nervous system.

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